Arthur S. Kunin

Decreased glycolytic enzyme activity in epiphyseal cartilage of cortisone-treated rats☆

Abstract Procedures are described for highly reproducible assays of protein content and the activity of hexokinase, phosphofructokinase, aldolase, pyruvate kinase, lactate dehydrogenase, NAD-linked glycerophosphate dehydrogenase, and glucose-6-phos-phate dehydrogenase in epiphyseal cartilage from rat tibiae. Glucokinase is not present. With cortisone treatment the four glycolytic enzymes, phosphofructokinase, aldolase, pyruvate kinase, and lactate dehydrogenase, exhibit a closely coordinated decrease in activity of over 50% per cell, or an average decrease of 34% in specific activity (Enzyme Units/milligram protein). Glycerophosphate dehydrogenase follows a generally similar pattern. Hexokinase and glucose-6-phosphate dehydrogenase are also decreased per cell, but only to the same extent as total protein so that there is little change in specific activity. Starvation brings about changes in the enzyme activity pattern similar to those of cortisone-treatment.

The inductive effect of rickets on glycolytic enzymes of rat epiphyseal cartilage and its reversal by vitamin D and phosphate

Abstract In rickets produced in rats by low phosphate, vitamin D-free diets, epiphyseal cartilage increases in wet weight, and in deoxyribonucleic acid, glycogen, and protein content. Dietary phosphate reverses all of these effects. Vitamin D reverses all of these rachitic changes with the exception of that in deoxyribonucleic acid content. The effectiveness of vitamin D in reducing cartilage cell hypertrophy without decreasing cell numbers differentiates the mechanism of vitamin D action from that of phosphate. Glycolysis is doubled per cell in rachitic cartilage and there is a corresponding coordinate increase in specific activities (EU/mg protein) of phosphofructokinase, aldolase, pyruvate kinase, and lactate dehydrogenase. Both effects are reversed by dietary phosphate or vitamin D. It is suggested that increased glycolysis may be due to a specific increment in glycolytic enzyme levels supplemented by a general increase in tissue protein. A proportional relationship between cellular lactate production and glycolytic enzyme activity is demonstrated in nine different physiological states. The hypothesis is advanced that a regulatory mechanism operates in cartilage to coordinately control the levels of glycolytic enzymes and cellular metabolism.

The effect of cortisone on the intermediary metabolism of epiphyseal cartilage from rats.

Abstract Total glucose stores per cell are reduced by half in tibial epiphyseal cartilage from rats after 3 days of cortisone treatment, and the ability to produce lactate in vitro without added substrate is abolished. Cortisone reduces lactate production from exogenous glucose to 30% of normal. The pentose shunt pathway and lactate formation from pyruvate also are decreased. In contrast, 14CO2 evolution from glucose-6-14C or labeled pyruvate is unaffected by cortisone treatment suggesting that the potential rate of pyruvate oxidation via the citric acid cycle is relatively insensitive to the hormone. High levels of pyruvate in vitro inhibit lactate formation from labeled glucose by over 90% in normal and cortisone-treated tissue while doubling 14CO2 evolution from glucose-1-14C. Total lactate production is depressed while glucose utilization is stimulated suggesting that pyruvate can inhibit glycolysis in cartilage tissue and divert glucose to alternate fates such as the pentose shunt and glycogen synthesis. The controlling influence of such hormonal and metabolic mechanisms on glycolysis may contribute significantly to the marked atrophic effect of glucocorticoids on growth cartilage as well as to the phenomenon of decreased peripheral glucose utilization induced by these hormones.

Effects of cortisone, starvation, and rickets on oxidative enzyme activities of epiphyseal cartilage from rats.

Abstract Epiphyseal cartilage fractions from rats have been shown to have the enzymatic complement for oxidizing a wide variety of substrates though at relatively low rates compared to tissues such as liver and heart. In contrast to previous data for glycolytic enzymes, mitochondrial oxidative enzyme levels do not appear to be specifically affected by dietary rickets, starvation, or cortisone treatment and do not correlate with the oxidative activity of cartilage slices. These findings give added emphasis to our earlier suggestion that control of glycolytic enzyme levels plays a central role in regulation of cartilage cell economy. A marked difference in the relative distribution between supernatant and pellet fractions of glycerol-3- P oxidase compared to other typical mitochondrial enzymes including succinate dehydrogenase is interpreted as evidence for two classes of mitochondria in cartilage. According to this hypothesis, there is a class of more readily sedimented mitochondria which contain relatively much more glycerol-3- P oxidase. Although this enzyme is thought to play a role in regulation of glycolysis, the control of synthetic-degradative mechanisms for it does not appear to be coordinated with those for the glycolytic enzymes and glycerol-3- P dehydrogenase of the cartilage cytoplasm. It is suggested that the oxidase may have a special role in Ca 2+ accumulation by mitochondria.

Effects of Hemodialysis and of Glucose-Insulin Administration on Plasma Potassium and on the Electrocardiogram

Hypopotassemia was produced in dogs by hemodialysis or by intravenous administration of glucose with insulin. Both methods resulted in a similar decrease in plasma K concentration and similar ECG changes although K was withdrawn from the cells during dialysis while it entered the cells during glucose-insulin infusion. The maximal plasma K decrease occurred after two hours of dialysis or glucose-insulin infusion; administration of glucose-insulin after dialysis resulted in no further plasma potassium decrease. However, dialysis after glucose-insulin infusion caused further lowering of plasma potassium. The ECG changes were evaluated in relation to changes in plasma K concentration. The most consistent effects of hypopotassemia were in decreasing order: increased duration of the QRS complex, increased duration of the QTc interval and increase in amplitude of the P wave. Correlation coefficients (R) between these ECG changes and the plasma K decreases were of the order −0.48 to −0.54. The ECG changes failed to show even a weak correlation with absolute plasma K concentrations or with calculated changes in intracellular K.

Intestinal calcium-binding protein and rickets.

Summary To determine the influence of dietary phosphate deficiency and rickets on the level of intestinal calcium-binding protein, weanling rats were raised on 2 types of vitamin D-deficient diets. A group fed a rachitogenic diet, which was vitamin D-free, high in calcium, and low in phosphorus, developed rickets as measured by increased lactate formation after in vitro incubation of tibial epiphyseal cartilage slices. A second group, fed the same diet supplemented with phosphate, did not develop rickets. The level of intestinal calcium-binding activity was the same in animals raised on both dietary regimens and vitamin D administration induced an increase in binding activity of the same magnitude in both groups of rats. Thus the synthesis of calcium-binding protein appears to be independent of dietary phosphate deficiency and of the presence of rickets.

Urate oxidation by cupric ion (Cu

Uric acid or urate, a compound of extremely limited solubility produced in vivo by degradation of purines from nucleic acids or other endogenous and exogenous sources has long been considered a metabolic “enfant terrible” of man. Possessing a vestigial uricotelic apparatus but lacking the copper-containing enzyme uricase which oxidizes urate to water-soluble compounds, man teters on the brink of urico-disaster. Although elaborate control mechanisms exist to prevent flooding by urate, when these systems fail for genetic or other reasons body urate pools increase and there is danger of precipitation in soft tissues, cartilage (tophi) and urine (stones). While the major portion (60-70%) of the urate disappearing from the urate pool is excreted as such in urine, the remainder may be converted to CO,, allantoin, allantoic acid, urea, and NH, by bacteria of the gastrointestinal tract [ 1 J . In addition oxygen uptake studies have revealed that urate may be oxidized by Cu++ above pH 10 and by the cytochrome oxidase system (Cu++?) at pH 7.9 [2]. Hemoproteins in the presence of peroxide causes breakdown of urate to allantoin, CO, and alloxanic acid [3]. We are reporting in vitro urate oxidation mediated solely by Cu++ at pH 6.0-8.2. In fig. 1 the fall in absorption of a urate solution incubated with Cu++ (urate/Cu++ ratio 1:4) is shown. Cu++ also alters the shape of the urate absorption spectrum drastically, obliterating the second maximum at 237 rnp. H,S

Intestinal mitochondrial calcium uptake during adaptation to dietary calcium restriction

In order to evaluate the role of mitochondrial calcium uptake in intestinal calcium absorption, the effect of adaptation to dietary calcium deficiency on the in vitro uptake of calcium by isolated duodenal mitochondria was studied. Experiments were performed utilizing 28-day-old cockerels which had received a diet adequate in vitamin D3 and phosphate and containing either 0.08% or 1.20% calcium. Mitochondrial45Ca uptake from chicks deprived of dietary calcium was not significantly different from controls. These results suggest that increased calcium uptake by intestinal mitochondria is not crucial for adaptation to a low calcium diet.

Bacteriuria in Sodium-Losing Nephritis

PATIENTS with renal disease occasionally lose the ability to conserve sodium adequately. Although first reported by Peters et al.1 in 1929, sodium-losing nephritis was not a well defined clinical s...

Effect of pH on metabolism of the glutamine carbon skeleton by renal cortical mitochondria

Abstract 1. 1. To determine the effect of altered acid-base homeostasis on the intramitochondrial metabolism of the glutamine carbon skeleton 14CO2 production from [U-14C]glutamine by isolated rat renal cortical mitochondria was measured. 2. 2. Mitochondria from rats with chronic metabolic acidosis either showed no change or diminished 14CO2 production in comparison with pair fed controls. 3. 3. By contrast, when the pH of the medium incubating mitochondria from normal rats was manipulated (pH 7.0, 7.4, 7.7), 14CO2 production was clearly altered, but the direction and magnitude of the change depended on the glutamine concentration used (0.5 or 10.0 mM). 4. 4. Mitochondria produced significant quantities of 14CO2 when [1,4 14C]succinate was used as substrate, indicating that 14CO2 production from glutamine does not originate solely from the decarboxylation of α KG. 5. 5. Thus chronic acidosis and pH, per se, affect intramitochondrial glutamine carbon skeleton metabolism in different fashions, but the specific mechanism cannot be elucidated using 14CO2 production from [U-14C]glutamine. 6. 6. Additional studies directly quantitating the metabolic products of glutamine have confirmed these findings and more precisely defined the sites of metabolic alteration.